PROJECT SUMMARY/ABSTRACT Given the improved efficacy of antiretroviral (ARV) therapy (ART), HIV-infected persons are living near-normal lifespans. Therefore, it is critical to understand the adverse consequences of long-term ART use. Our previous clinico-pathological study using samples from the National NeuroAIDS Tissue Consortium (NNTC) demonstrated direct association between protease inhibitor-based ART and higher risk of cerebral arteriolosclerosis. This has important clinical implication because mild arteriolosclerosis was found to associate with neurocognitive impairment even after adjusting for HIV encephalitis or older age. We hypothesize that ARV-associated arteriolosclerosis may be mediated by the acceleration of vascular cell senescence related to advanced glycation end products (AGEs) and receptor for AGEs (RAGE) signaling. The early stages of small vessel degeneration are characterized by vascular cell degeneration and basement membrane thickening. We propose a set of novel approaches to 1) examine the relationship between ART and early-stage premature degeneration of small vessels in the brain (using NNTC samples), 2) identify small vessel degeneration biomarkers in the cerebrospinal fluid (CSF), and 3) explore cellular mechanisms underlying small vessel degeneration using 3D co-cultures of human primary brain vascular cells. In Aim 1, we will study the association of early-stage small vessel degeneration in the frontal white matter with ART. 1.1) We will measure immunoreactivity in uninterrupted small vessel segments for AGEs, RAGE, and markers of cell senescence, vascular cell types, and basement membranes in optically transparent 1,000-m-thick brain sections, using a cutting-edge tissue clearing technique and fluorescence microscopy for 3D proteomic imaging and quantitative phenotyping. 1.2) We will assess vessel homogenates isolated from the adjacent brain tissue for transcript and protein expression of the same set of markers. In Aim 2, to identify CSF biomarkers of early-stage vascular degeneration, we will assay CSF samples for soluble forms of the protein markers examined in Aim 1. In Aim 3, we will explore in vitro the role of the AGEs-RAGE axis in ARV-induced vascular cell senescence. Vascular endothelial cells, smooth muscle cells, and pericytes will be co-cultured in 3D multi-layer scaffold systems (mimicking the in vivo small vessel wall). We will assess in cultured cells changes of AGEs, RAGE, cell senescence-associated markers, and cell type-specific proteins. In conditioned media (mimicking the CSF), soluble forms of the proteins will be measured. For ARV drugs showing cytotoxic effects, the intracellular AGEs-RAGE signaling pathways will be explored in vascular cell co-cultures. This translational research is consistent with the research areas supported by the NINDS and included in the 2017 Overarching HIV/AIDS Research Priorities for AIDS Funding, specifically premature aging associated with long-term ART. In future studies, we plan to test the usefulness of the CSF biomarkers in living patients on long-term ART who are at risk for cerebral small vessel disease and neurocognitive impairment.